EPHRAIM AND OTHERS

NOVEL PORTABLE MICROSCOPY FOR SCHISTOSOMIASIS DIAGNOSIS

Diagnosis of Schistosoma haematobium Infection with a Mobile Phone-Mounted

Foldscope and a Reversed-Lens CellScope in Ghana

Richard K. D. Ephraim, Evans Duah, James S. Cybulski, Manu Prakash, Michael V.

D’Ambrosio, Daniel A. Fletcher, Jennifer Keiser, Jason R. Andrews,† and Isaac I. Bogoch*†

Division of Medical Laboratory Technology, University of Cape Coast, Cape Coast, Ghana; Department of

Mechanical Engineering, Stanford University, Stanford, California; Department of Bioengineering, Stanford

University, Stanford, California; Department of Bioengineering, University of California, Berkeley, Berkeley,

California; Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute,

Basel, Switzerland; University of Basel, Basel, Switzerland; Division of Infectious Diseases and Geographic

Medicine, Stanford University School of Medicine, Stanford, California; Divisions of Internal Medicine and

Infectious Diseases, University Health Network, Toronto, Ontario, Canada; Department of Medicine, University of

Toronto, Toronto, Ontario, Canada

* Address correspondence to Isaac I. Bogoch, Divisions of Internal Medicine and Infectious Diseases, Toronto General Hospital,

14EN- 209, 200 Elizabeth Street, Toronto, ON, Canada M5G 2C4. E-mail: isaac.bogoch@uhn.ca

† These authors contributed equally to this work.

Abstract.

We evaluated two novel, portable microscopes and locally acquired, single-ply, paper towels as filter paper for the

diagnosis of Schistosoma haematobium infection. The mobile phone-mounted Foldscope and reversed-lens

CellScope had sensitivities of 55.9% and 67.6%, and specificities of 93.3% and 100.0%, respectively, compared

with conventional light microscopy for diagnosing S. haematobium infection. With conventional light microscopy,

urine filtration using single-ply paper towels as filter paper showed a sensitivity of 67.6% and specificity of 80.0%

compared with centrifugation for the diagnosis of S. haematobium infection. With future improvements to

diagnostic sensitivity, newer generation handheld and mobile phone microscopes may be valuable tools for global

health applications.

INTRODUCTION

Microscopy is an integral tool of clinical medicine and public health, however this basic

technology is not routinely available in health centers in resource-constrained settings.1 Many

individuals live in rural or underserviced locations with limited access to care, and where

infections such as malaria, schistosomiasis, and soil-transmitted helminths are rife.2,3

Handheld microscopes,4–6

and more recently, mobile phone-based microscopes7,8

have been

used in field settings for the diagnosis of malaria, schistosomiasis, and soil-transmitted

helminthiasis. These devices have the benefit of being portable and relatively simple to use.

Other recent innovations that support inexpensive diagnostic testing in underserviced locations

include using locally procured paper products such as paper towels as low-cost filters for the

diagnosis of Schistosoma haematobium infections.9 Such innovations hold promise for delivering

low-cost laboratory-based technology to underserviced locations, and may have use in clinical

and public health settings.

In order to provide our readers with timely access to new content, papers accepted by the American Journal of Tropical Medicine and Hygiene are posted

online ahead of print publication. Papers that have been accepted for publication are peer-reviewed and copy edited but do not incorporate all corrections or

constitute the final versions that will appear in the Journal. Final, corrected papers will be published online concurrent with the release of the print issue.

http://ajtmh.org/cgi/doi/10.4269/ajtmh.14-0741The latest version is at Accepted for Publication, Published online April 27, 2015; doi:10.4269/ajtmh.14-0741.

Copyright 2015 by the American Society of Tropical Medicine and Hygiene

Here, we evaluate two novel handheld and mobile phone-based microscopes and

inexpensive, locally acquired paper towels as filter paper for the diagnosis of S. haematobium

infections in a rural Ghanaian community.

METHODS

This study was integrated into an ongoing epidemiologic survey of schistosomiasis and soil-

transmitted helminthiasis in the Central Region of Ghana between February and May 2014. The

University of Cape Coast (Cape Coast, Ghana) granted ethical permission for this project.

Headmasters at participating schools, pupils, and their parents consented for involvement in this

study. For the purpose of this sub-study, we randomly selected 50 individuals enrolled in a

participating school with 200 students 7–13 years of age in Sorodofo-Abaasa village, in the

Abura Asebu Kwamankese district in the Central Region of Ghana, an area known to be endemic

for S. haematobium. A urine sample from each participant was collected between 10:00 AM and

14:00 PM,10

and processed on the same day of collection at the University of Cape Coast Hospital

Laboratory. Urine samples were shaken and 20 mL was extracted by syringe, with 10 mL

processed by centrifugation (as gold standard) and 10 mL by the experimental gravity filtration

approach. The centrifuged urine was spun at 5,000 rpm for 5 minutes. The supernatant was

discarded and sediment was transferred to a glass microscope slide with the addition of a

coverslip. The other 10 mL of urine extracted was gravity filtered through single-ply paper

towels that were purchased locally. The methods of this procedure are outlined elsewhere.9

Briefly, the paper towel was formed into a cone, and urine was poured into the center of the cone

and allowed to filter through. The central portion of the paper towel where urine was poured

through was cut out and placed directly onto a glass microscope slide. Conventional light

microscopy (using 10 and 20 objective lenses) with a CX21LEDFS1 Olympus microscope

(Olympus, Volketswil, Switzerland), and two novel handheld light microscope devices evaluated

both centrifuged and filtered urine. Expert microscopists who were blinded to prior S.

haematobium ova counts on each slide performed microscopy. The entire slide was evaluated for

a minimum of 3 minutes. The presence or absence of ova was noted, and if present was

quantified under conventional light microscopy. We only documented the presence or absence of

ova with novel light microscopes. All data were directly entered into an Excel file (Microsoft

Corp., Redmond, WA).

Experimental microscopes included the mobile phone-mounted Foldscope11

and a reversed-

lens CellScope.12

The Foldscope is a small, handheld, paper-based microscope with a light-

emitting diode (LED) light source. It can be held up to the eye for visualization of slides,

however we secured it to the camera lens of an iPhone 5S (Apple, Cupertino, CA) by tape and

magnets (Figure 1A). Microscope slides were manually manipulated under the phone-mounted

Foldscope lens, and the presence or absence of ova was noted on the iPhone screen. The

CellScope is a mobile phone microscope that integrates imaging and illumination optics directly

with an unmodified mobile phone.13,14

The reversed-lens CellScope12

(Figure 1B) used in this

study is a small three-dimensional-printed plastic attachment for the iPhone 5S with an

embedded lens for microscopic imaging. It harnesses the mobile phone’s light source to

illuminate slides by reflecting light off of a white slide holder placed underneath the microscope

slide. The lens embedded in the attachment aligns with the iPhone camera lens, and a magnified

portion of the slide can be visualized on the screen of the mobile phone using the camera

function. A microscopist held the device directly above a microscope slide and manually

manipulated the device across the full slide, while examining the mobile phone screen for the

presence or absence of S. haematobium ova.

Using the same slides prepared by urine centrifugation, we measured the sensitivity and

specificity of the two portable microscopes using conventional light microscopy as the gold

standard, and compared dichotomous agreement using Cohen’s Kappa. We similarly examined

sensitivity, specificity, and agreement of urine filtration with centrifugation, using light

microscopy. All analyses were performed using R (R Foundation for Statistical Computing,

Vienna, Austria).

RESULTS

One urine sample was lost during laboratory processing, leaving 49 urine samples for

evaluation. Based on gold standard conventional light microscopic examination by

centrifugation, 34 of the 49 urine samples were positive for S. haematobium ova (mean: 8.7

eggs/10 mL), and of those infected, all but one was light intensity infected (50 eggs/10 mL).15

When evaluating centrifuged urine samples prepared on a microscope slide, the mobile phone-

mounted Foldscope and reversed-lens CellScope showed a sensitivity of 55.9% (95% confidence

interval [CI]: 38.1–72.4%) and 67.6% (95% CI: 49.4–82.0%), respectively, and a specificity of

93.3% (66.0–99.7%) and 100.0% (74.7–100.0%), respectively (Table 1). Images of S.

haematobium ova captured by each device can be viewed in Figure 1C and D. Agreement

between conventional microscopy and the phone-mounted Foldscope (= 0.39, 95% CI: 0.18–

0.60) was fair, and agreement between conventional microscopy and reversed-lens CellScope

(= 0.56, 95% CI: [0.36–0.77]) was moderate. Neither device could reliably detect S.

haematobium ova on slides prepared by gravity filtration through single-ply paper towels, and

hence were not quantified.

Gravity filtration of urine samples through single-ply paper towels and examined with

conventional microscopy showed sensitivity of 67.6% (95% CI: 49.4–82.0%), specificity of

80.0% (51.4–94.7%), and fair to moderate correlation (= 0.41, 95% CI: 0.17–0.66) compared

with slides prepared with centrifuged urine (Table 1). Schistosoma haematobium ova centrifuged

or filtered through single-ply paper towel and examined with conventional microscopy can be

viewed in Figure 1E and F, respectively.

DISCUSSION

Robust, simple, and inexpensive diagnostic tests are greatly needed in resource-constrained

settings. Handheld and mobile phone-based microscopes may have an important role in

providing such diagnostic support.13,16

In this work we studied two novel, handheld, mobile-

phone compatible microscopes and single-ply paper towels to filter urine for the diagnosis of S.

haematobium in individuals with low-intensity infections in an endemic setting.

The mobile phone-mounted Foldscope11

had limited sensitivity, but excellent specificity for

the diagnosis of S. haematobium infection. This device, which uses a 2.38 mm ball lens for

magnification and is illuminated by a small battery powered LED, was effective at focusing on

ova when in the field of view. The Foldscope weighs < 8 g, costs < 1 United States Dollar

(USD). The sensitivity of this device may be low because of a combination of some challenges

with manually manipulating microscope slides underneath the lens, and the low infection

intensities in the study setting. The Foldscope used in this project was able to magnify images by

140, however other greater power magnifications are available as well. Based on preliminary

trials, we felt the 140 magnification was easiest to use, and when coupled with the digital zoom

of the mobile phone camera would provide adequate magnification for S. haematobium ova.

The reversed-lens CellScope12

showed modest sensitivity but excellent specificity for S.

haematobium diagnosis in this study. As with the Foldscope, the limited sensitivity may be

caused by our manual manipulation of the device over slides with very light intensity infections.

The device was easy to handle and did not have issues with sample contamination from the

microscope slide. The reversed-lens CellScope weighs 5 g, uses a wide-field lens that costs < 6

USD, and provides 5 µm resolution over an 10 mm2 field of view.

12 When coupled with the

optical zoom function of the mobile phone camera, the device can provide adequate screen

magnification for S. haematobium ova identification.

These two novel devices show early promise and may be a stepping stone for future portable

diagnostic devices. They could be used in clinical and epidemiologic settings in the near future

with technical adjustments to increase diagnostic sensitivity, such as increasing the field of view

and enabling magnification of different powers, in addition to validating these devices on other

pathogens.

Locally procured single-ply paper towel did not have adequate sensitivity for S.

haematobium diagnosis when evaluated by conventional light microscopy. Furthermore, we

could not detect S. haematobium ova on filter paper with the mobile phone-mounted Foldscope

or reversed-lens CellScope in the configurations used in this study, possibly because of how light

scattered off of the corrugated surface of the paper when illuminated from above. Early results

using inexpensive, locally procured filter paper evaluated by conventional light microscopy were

encouraging,9 however it appears that this type of paper is not suitable for routine laboratory use.

Schistosoma haematobium ova may have passed through larger pores in the paper or may have

been aligned in vertical or diagonal positions caused by the corrugated surface of the paper.

Microscopists may not have counted S. haematobium ova in these unusual positions. Single-ply

filter paper is strong and easy to manipulate when wet and it does not tear easily when

transferred to a microscope slide. Inexpensive filter paper used in future studies should have

these positive attributes but will need to have a smoother surface to reduce light refraction, while

ensuring pores are small enough such that ova cannot readily pass through.

Other recent innovations may aid in the field diagnosis of S. haematobium infection as well,

including a low-cost Millipore filtration device,17

or the more technologically advanced on-chip

imaging of ova,18

both of which showed promise in early proof-of-concept studies. However, all

will require vigorous field testing in clinical and public health settings before implementation.

Our study was limited by a few factors. We only detected the presence or absence of S.

haematobium ova with experimental microscopes and did not quantify infection intensity. Future

studies should quantify ova counts, and also evaluate a cohort where there are both high and low

intensity infections. In addition, laboratory technicians rather than expert microscopists should

be included in evaluating slides to more closely proxy real-world settings.

Both the mobile phone-mounted Foldscope and reversed-lens CellScope have excellent

specificity for diagnosing S. haematobium in those with light intensity infections in an endemic

setting. With future modifications to improve sensitivity, such devices may be useful diagnostic

tools in resource-constrained clinical and public health settings.

Received November 22, 2014.

Accepted for publication January 21, 2015.

Financial support: Isaac Bogoch is supported by Grand Challenges Canada.

Authors’ addresses: Richard K. D. Ephraim and Evans Duah, Division of Medical Laboratory Technology,

University of Cape Coast, Cape Coast, Ghana, E-mails: kdephraim@yahoo.com and ivanprecy@gmail.com. James

S. Cybulski and Manu Prakash, Department of Mechanical Engineering, Stanford University, Stanford, CA, and

Department of Bioengineering, Stanford University, Stanford, CA, E-mails: cybulski@stanford.edu and

manup@stanford.edu. Michael V. D’Ambrosio and Daniel A. Fletcher, Department of Bioengineering, University of

California, Berkeley, CA, E-mails: mdambrosio@berkeley.edu and fletch@berkeley.edu. Jennifer Keiser,

Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel,

Switzerland, and University of Basel, Basel, Switzerland, E-mail: jennifer.keiser@unibas.ch. Jason R. Andrews,

Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA,

E-mail: jandr@stanford.edu. Isaac I. Bogoch, Divisions of Internal Medicine and Infectious Diseases, Toronto

General Hospital, Toronto, ON, Canada, E-mail: isaac.bogoch@uhn.ca.

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FIGURE 1. A mobile phone-mounted Foldscope (A), and reversed-lens CellScope (B). Schistosoma haematobium

ova visualized by a mobile phone-mounted Foldscope after centrifugation (C), reversed-lens CellScope after

centrifugation (D), conventional microscopy after centrifugation (E), and conventional microscopy after filtration

with single-ply paper towel (F). Reference bars = 100 m. This figure appears in color at ajtmh.org.

TABLE 1

Sensitivity, specificity, and Kappa comparing conventional light microscopy with single-ply paper towels to urine

centrifugation, and conventional light microscopy to the mobile phone-mounted Foldscope and reversed-lens mobile

phone microscope for the diagnosis of Schistosoma haematobium infection

Single-ply filter paper Phone-mounted Foldscope Reversed-lens CellScope

Negative Positive Negative Positive Negative Positive

Urine centrifugation and

conventional light microscopy

Negative 12 3 14 1 15 0

Positive 11 23 15 19 11 23

Kappa 0.41, 95% CI (0.17–0.66) 0.39, 95% CI (0.18–0.60) 0.56, 95% CI (0.36–0.77)

Sensitivity 0.68 0.56 0.68

Specificity 0.80 0.93 1.000

CI = confidence interval.

Figure 1